The diminished EOG light peak is believed to be caused by a defective Ca2
+-activated Cl
− channel in the basolateral membrane of the retinal pigment epithelium (RPE), where bestrophin is expressed.
10 The disruption of ion flow across the membrane due to
BEST1 mutations may explain the pathologic changes demonstrated in the pathologic EOG Arden ratio in all but two patients in the present study. If so, since bestrophin is expressed throughout the RPE, one might expect a correlation between the magnitude of the Arden ratio and the total retinal function; however, this was not the case. Furthermore, most patients had a normal ffERG. A disproportionate reduction of the EOG compared to the ffERG indicates a primary defect in the function of the RPE. The generalized retinal degeneration shown by a reduced ffERG in patients with biallelic
BEST1 mutations—for example, in families SI and DII—may result from a mechanism different from the one that causes macular degeneration.
A reduced EOG Arden ratio is considered to be the most penetrant clinical sign of BMD. In the present study, all patients except for SI I:2 (heterozygous for p.Arg141His) and DIV I:2 (heterozygous for p.Leu82Val) had clearly reduced EOG Arden ratios. Patient DIV I:2 demonstrated a borderline reduction of the Arden ratio, and clinical signs including mfERG, OCT, and FAF compatible with BMD. Moreover, the patients' sister harbored the same mutation, and had a reduced EOG Arden ratio (
Table 2). The mutation was also described previously in a patient with BMD in a study that included EOG; however, EOG details were not given for this particular patient.
17
Regarding p.Arg141His, a normal EOG was similarly described in one clinically unaffected individual heterozygous for this mutation, whose daughter carried the compound heterozygous mutations p.Leu41Pro/p.Arg141His in
BEST1 and was affected with ARB.
4 Thus, the mutation may not be penetrant in the EOG Arden ratio and seems to cause disease only in a compound heterozygous or recessive mode.
In this study, we found a novel mutation in exon 8, c.936C>A leading to p.Asp312Glu. The mutation is located in a conserved protein region (
Fig. 2B) and several BMD-causing mutations have been reported to affect adjacent amino acids at positions 292 to 311 (see Sohn et al.
3 for a report regarding the p.Glu292Lys mutation and a review by White et al.
18 ). A mutation causing adult-onset VMD and affecting the same amino acid, p.Asp312Asn, has been reported previously.
19 The latter was also reported as a compound heterozygous mutation in association with autosomal recessive bestrophinopathy, together with the mutation p.Met325Thr.
4 In addition, in a recent report, mutations affecting the N terminus (p.Arg19Cys, p.Arg25Cys, and p.Lys30Cys) and the C terminus (p.Gly299Glu, p.Asp301Asn, and p.Asp312Asn) caused channel dysfunction, probably resulting from disruption of the N and C terminus interaction.
20 We therefore conclude that p.Asp312Glu is a probable disease-causing mutation. Consanguineous marriages in this family led to a rare situation in which a patient was homozygous for a disease-causing mutation. Of interest, the patient presented with a multifocal VMD and evidence of widespread retinal degeneration as demonstrated by ffERG. The heterozygous father of the homozygous boy, however, demonstrated atypical Bull's eye–like central atrophy in both eyes. The heterozygous father presented with ffERG responses within normal limits and had a clearly reduced EOG Arden ratio, compatible with AD BMD, which provides further support for the notion that this novel mutation is pathogenic.
Histopathology from a patient homozygous for the dominant mutation p.Trp93Cys and another patient heterozygous for the mutation p.Thr6Arg demonstrated a similar increase in the retinal pigment epithelium content of A2E and lipofuscin in both patients. The homozygous patient did not demonstrate any more severe structural alterations than the heterozygous patient.
21 This finding seems to differ from those in a homozygous patient in the present study who demonstrated a multifocal vitelliform dystrophy and had signs of widespread retinal degeneration featuring a reduced ffERG rod response. A similar reduction of ffERG rod response was reported previously in ARB.
4
Different mutations may cause disease by different mechanisms. A functional analysis of an exon 8 variant (p.Gln293His) in human embryonic kidney cells revealed a severe reduction of chloride current that behaved in a dominant negative manner, inhibiting the function of wild-type bestrophin-1 channels.
22 In the present study, the finding of a homozygous dominant mutation in a patient DII II:1 with vitelliform macular dystrophy and evidence of widespread retinal degeneration, may indicate that the pathogenesis of the macular degeneration differs from that of the generalized retinal degeneration.
Delayed implicit time in the ffERG 30-Hz cone flicker, indicating widespread retinal degeneration, was seen in two sisters from family SI aged 30 and 33 years, who harbored compound heterozygous mutations in
BEST1.
7 The genetic alteration in the former seems to closely resemble ARB and those found in canine multifocal retinopathy, which has been described as a recessive disease, with the mutations p.Arg25X and p.Gly161Asp in
best1.
4,8 In ARB, patch–clamp studies showed a reduced channel function that was restored after cotransfection with wild-type bestrophin, consistent with a loss-of (channel)-function mechanism of disease.
4 However, biological events other than regulation of ion flow in the retinal pigment epithelium, such as ceramide accumulation, may be involved in the bestrophin-associated disease process.
12
Based on the findings in the present study and previous studies, it seems that compound heterozygous, biallelic recessive or homozygous dominant mutations in
BEST1 may confer a particularly severe phenotype, featuring widespread retinal degeneration, in addition to VMD.
4,7 On the other hand, in dominant heterozygous BMD, the variable phenotype is again highlighted in the present study, and there seems to be no clear pattern relating type of
BEST1 mutation to severity of clinical expression. For example, in family DIII with the heterozygous
BEST1 mutation c.275G>A leading p.Arg92His, a 6-year-old boy presented with early-onset VMD, including reduced vision and thickening of the outer retina, whereas the only significant finding in the father of the boy was a reduced EOG Arden ratio (
Fig 4). Similar intrafamilial phenotypic variability was demonstrated, for example, in family DI, with the heterozygous mutation c.253T>C leading to p.Tyr85His and in family SII with the heterozygous mutation c.266T>C leading to p.Val89Ala. On the other hand, a bilateral multifocal VMD was seen in at least four of eight individuals with the former genotype (c.253T>C leading to p.Tyr85His;
Table 1,
2;
Figs. 3,
4). Thus, multifocal VMD may be overrepresented in this genotype. The severity of the novel mutation in exon 8, c.936C>A leading to p.Asp312Glu, found in the large consanguineous family DII remains to be determined, as only one heterozygous individual was available for examination.
mfERG typically revealed amplitude reduction in the central macular area and preserved function in the periphery (
Figs. 3,
4). This finding has been reported previously in BMD, and may be a feature in common with other disorders affecting primarily the retinal pigment epithelium, such as Bothnia dystrophy.
23 However, a few patients had preserved mfERG responses, normal visual acuities, and normal OCT scans, consistent with variable expression and penetrance of disease phenotype. mfERG amplitudes were significantly correlated to VA, which is in line with previous results.
24 mfERG implicit times, however, were delayed in the periphery in several patients, indicating a subtle dysfunction, as well in the periphery, in addition to the central retinal dysfunction. A significant correlation between age and VA has been described in BMD, with most patients younger than 40 years having VA > 20/40.
13 In this study, however, a few young patients had significant visual disability (
Fig. 9).
mfERG implicit times may be a sensitive tool for detecting early functional disturbances in BMD, as has been suggested in diabetic retinopathy and Stargardt disease, in which implicit time delays may predict future sites of retinopathy.
25–27 In patients with multifocal distribution of hyperautofluorescence by FAF, peripheral mfERG implicit times were delayed (
Figs. 4,
11). In a previous study on mfERG responses in BMD, only a slight but significant delay was noted for peripheral, but not central, implicit times.
24 In the latter, a different form of mfERG was used featuring only 61 hexagonal stimulus elements, compared with 103 in our study, with a decrease in resolution of the responses.
Different patterns were identified with OCT, according to clinical stage. Some of these, including thickening of the ORCC and subretinal fluid, have been described earlier in BMD.
7,28 The thickened hyperreflective ORCC would probably correspond to the accumulation of lipofuscin and A2E in and beneath the RPE, as has been shown in histopathologic studies of BMD.
21,29 This finding is also supported by the hyperautofluorescence in fundus autofluorescence photography. The accumulation of lipofuscin and A2E is probably one of the primary steps in the pathogenesis of retinal degeneration and visual loss. Despite this pattern on OCT, (e.g., III:1 in family SII p.Val89Ala,
Table 2,
Fig. 5) VA and central retinal function can still be preserved on mfERG. Recently, however, it has been suggested that a supranormal mfERG is a sign of impending visual loss (Koozekanani DD, et al.
IOVS 2006;47:ARVO E-Abstract 3754).
Fundus autofluorescence photography is a sensitive method of revealing lipofuscin-containing material in various degenerative retinal conditions, including BMD.
3,17,30 The distribution of hyperautofluorescence may extend the funduscopically visible changes, as in patient DI I:1, heterozygous for the mutation p.Tyr85His and in the 9-year-old boy homozygous for the mutation c.936C>A leading to p.Asp312Glu. We have recently shown that patients with fundus albipunctatus due to mutations in
RDH5 and hence an inhibition of the retinoid cycle leading to diminished A2E formation have a generalized homogenous reduction of fundus autofluorescence, implying reduced lipofuscin formation. These patients may have relatively well-preserved retinal function,
31 which implies a potential treatment for vitelliform dystrophy and BMD: a pharmacologic inhibition of the retinoid cycle.
32 If so, high-resolution OCT findings—for example, demonstration of preserved photoreceptors and absence of atrophy and fibrosis—may indicate eligibility for treatment.
Central foveal thickness, as measured with OCT, did not significantly correlate with VA in our study. This finding is similar to retinitis pigmentosa, but in contrast to diabetic retinopathy, where retinal function seems to deteriorate when foveal retinal thickness exceeds 280 μm.
33,34 In Stargardt macular degeneration, a statistically significant association between central foveal thickness and VA has been demonstrated, reflecting the pathogenesis of visual loss in this form of macular degeneration, which seems to be related to progressive central retinal atrophy.
35 In BMD, the pathogenesis is more complex and dynamic, where different types of structural alterations, including thickening of the ORCC and subretinal fluid/retinal edema, precede the end-stage atrophy, thus making it difficult to relate simply foveal thickness to function. In this context, we should mention that the OCT protocol differed among Danish and Swedish patients, which may be a source of bias when seeking to compare retinal thickness among patients. To reduce dissimilarities due to differences in methodology, retinal thickness was measured with calipers in all patients, thus avoiding any bias due to different automated software procedures measuring retinal thickness. Even so, we cannot exclude a systematic bias involved. However, since a comparison between the two groups was not of interest, only a possible association between thickness and VA in the total cohort, this seems to further reduce the influence of a potential bias.
Furthermore, by OCT-4 which was performed in all Danish patients, a preserved integrity of the foveal photoreceptor IS/OS junction was related to VA, implying a secondary degeneration of foveal photoreceptors in VMD. Similar OCT findings were demonstrated in a previous study in VMD where
BEST1 mutations were reported in only a minority of patients.
36
To conclude, in vitelliform macular dystrophy associated with mutations in BEST1, the combination of molecular genetics, electrophysiology, and OCT is useful in assessment of diagnosis and disease severity. These methods may also provide a suitable tool for evaluating possible treatment outcomes in the future.
Supported by grants from the Swedish Society of Medicine, Dag Lenards fond, Stiftelsen för synskadade i f d Malmöhus län, Stiftelsen Kronprincessan Margaretas arbetsnämnd för synskadade, the Skane County Research and Development Fund, The Velux Foundation, The John and Birthe Meyer Foundation, the Danish Research Council, and Grant 3000003241 from the Chief Scientist Office of the Ministry of Health, Israel.